Cell cycle phase markers

ABSTRACT

The present invention relates to polypeptide and nucleic acids constructs which are useful for determining the cell cycle status of a mammalian cell. Host cells transfected with these nucleic acid constructs can be used to determine the effects that test agents have upon the mammalian cell cycle.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/572,510 filed Jan. 23, 2007, now U.S. Pat. No. 7,612,189, which is afiling under 35 U.S.C. §371 and claims priority to international patentapplication number PCT/GB2005/002884 filed Jul. 22, 2005, published onJan. 26, 2006, as WO 2006/008542, which claims priority to U.S.provisional patent application Nos. 60/590,814 filed Jul. 23, 2004,60/645,915 filed Jan. 21, 2005 and 60/645,968 filed Jan. 21, 2005.

GOVERNMENT SUPPORT

This invention was made with government support under GM052948 awardedby the NIH. The government has certain rights in the invention.

TECHNICAL FIELD

The present invention relates to cell cycle phase-specific markers andmethods for determining the transition between different phases of thecell cycle in mammalian cells.

BACKGROUND OF THE INVENTION

Eukaryotic cell division proceeds through a highly regulated cell cyclecomprising consecutive phases termed G1, S, G2 and M. Disruption of thecell cycle or cell cycle control can result in cellular abnormalities ordisease states such as cancer which arise from multiple genetic changesthat transform growth-limited cells into highly invasive cells that areunresponsive to normal control of growth. Transition of normal cellsinto cancer cells can arise though loss of correct function in DNAreplication and DNA repair mechanisms. All dividing cells are subject toa number of control mechanisms, known as cell-cycle checkpoints, whichmaintain genomic integrity by arresting or inducing destruction ofaberrant cells. Investigation of cell cycle progression and control isconsequently of significant interest in designing anticancer drugs(Flatt, P. M. and Pietenpol, J. A. Drug Metab. Rev., (2000), 32(3-4),283-305; Buolamwini, J. K. Current Pharmaceutical Design, (2000), 6,379-392).

Cell cycle progression is tightly regulated by defined temporal andspatial expression, localisation and destruction of a number of cellcycle regulators which exhibit highly dynamic behaviour during the cellcycle (Pines, J., Nature Cell Biology, (1999), 1, E73-E79). For example,at specific cell cycle stages some proteins translocate from the nucleusto the cytoplasm, or vice versa, and some are rapidly degraded. Fordetails of known cell cycle control components and interactions, seeKohn, Molecular Biology of the Cell (1999), 10, 2703-2734.

Accurate determination of cell cycle status is a key requirement forinvestigating cellular processes that affect the cell cycle or aredependent on cell cycle position. Such measurements are particularlyvital in drug screening applications where:

-   i) substances which directly or indirectly modify cell cycle    progression are desired, for example, for investigation as potential    anti-cancer treatments;-   ii) drug candidates are to be checked for unwanted effects on cell    cycle progression; and/or-   iii) it is suspected that an agent is active or inactive towards    cells in a particular phase of the cell cycle.

Traditionally, cell cycle status for cell populations has beendetermined by flow cytometry using fluorescent dyes which stain the DNAcontent of cell nuclei (Barlogie, B. et al, Cancer Res., (1983), 43(9),3982-97). Flow cytometry yields quantitative information on the DNAcontent of cells and hence allows determination of the relative numbersof cells in the G1, S and G2+M phases of the cell cycle. However, thisanalysis is a destructive non-dynamic process and requires serialsampling of a population to determine cell cycle status with time. Afurther disadvantage of flow cytometry techniques relates to theindirect and inferred assignment of cell cycle position of cells basedon DNA content. Since the DNA content of cell nuclei varies through thecell cycle in a reasonably predictable fashion, ie. cells in G2 or Mhave twice the DNA content of cells in G1, and cells undergoing DNAsynthesis in S phase have an intermediate amount of DNA, it is possibleto monitor the relative distribution of cells between different phasesof the cell cycle. However, the technique does not allow precision indetermining the cell cycle position of any individual cell due toambiguity in assigning cells to G2 or M phases and to furtherimprecision arising from inherent variation in DNA content from cell tocell within a population which can preclude precise discriminationbetween cells which are close to the boundary between adjacent phases ofthe cell cycle. Additionally, variations in DNA content and DNA stainingbetween different cell types from different tissues or organisms requirethat the technique is optimised for each cell type, and can complicatedirect comparisons of data between cell types or between experiments(Herman, Cancer (1992), 69(6), 1553-1556). Flow cytometry is thereforesuitable for examining the overall cell cycle distribution of cellswithin a population, but cannot be used to monitor the precise cellcycle status of an individual cell over time.

EP 798386 describes a method for the analysis of the cell cycle of cellsub-populations present in heterogeneous cell samples. This method usessequential incubation of the sample with fluorescently labelledmonoclonal antibodies to identify specific cell types and a fluorochromethat specifically binds to nucleic acids. This permits determination ofthe cell cycle distribution of sub-populations of cells present in thesample. However, as this method utilises flow cytometry, it yields onlynon-dynamic data and requires serial measurements to be performed onseparate samples of cells to determine variations in the cell cyclestatus of a cell population with time following exposure to an agentunder investigation for effects on cell cycle progression.

A number of researchers have studied the cell cycle using traditionalreporter enzymes that require the cells to be fixed or lysed. Forexample Hauser & Bauer (Plant and Soil, (2000), 226, 1-10) usedβ-glucuronidase (GUS) to study cell division in a plant meristem andBrandeis & Hunt (EMBO J., (1996), 15, 5280-5289) used chloramphenicalacetyl transferase (CAT) fusion proteins to study variations in cyclinlevels. U.S. Pat. No. 6,048,693 describes a method for screening forcompounds affecting cell cycle regulatory proteins, wherein expressionof a reporter gene is linked to control elements which are acted on bycyclins or other cell cycle control proteins. In this method, temporalexpression of a reporter gene product is driven in a cell cycle specificfashion and compounds acting on one or more cell cycle controlcomponents may increase or decrease expression levels.

U.S. Pat. No. 6,159,691 describes nuclear localisation signals (NLS)derived from the cell cycle phase-specific transcription factors DP-3and E2F-1 and claims a method for assaying for putative regulators ofcell cycle progression. In this method, nuclear localisation signals(NLS) derived from the cell cycle phase specific transcription factorsDP-3 and E2F-1 may be used to assay the activity of compounds which actto increase or decrease nuclear localisation of specific NLS sequencesfrom DP-3 and E2F-1 fused to a detectable marker.

Jones et al (Nat Biotech., (2004), 23, 306-312) describe a fluorescentbiosensor of mitosis based on a plasma membrane targeting signal and anSV40 large T antigen NLS fused to EYFP. Throughout the cell cycle thereporter resides in the nucleus but translocates to the plasma membraneduring mitosis, between nuclear envelope breakdown and re-formation.

WO 03/031612 describes DNA reporter constructs and methods fordetermining the cell cycle position of living mammalian cells by meansof cell cycle phase-specific expression control elements and destructioncontrol elements.

Gu et al. (Mol Biol Cell., 2004,15, 3320-3332) have recentlyinvestigated the function of human DNA helicase B (HDHB) and shown thatit is primarily nuclear in G1 and cytoplasmic in S and G2 phases, thatit resides in nuclear foci induced by DNA damage, that the focal patternrequires HDHB activity, and that HDHB localization is regulated by CDKphosphorylation.

None of the preceding methods specifically describe sensors which can bestably integrated into the genome and used to indicate G1, S and G2phases of the cell cycle. Consequently, methods are required that enablethese phases of the cell cycle to be determined non-destructively in asingle living mammalian cell, allowing the same cell to be repeatedlyinterrogated over time, and which enable the study of the effects ofagents having potentially desired or undesired effects on the cellcycle. Methods are also required that permit the parallel assessment ofthese effects for a plurality of agents.

SUMMARY OF THE INVENTION

The present invention describes a method which utilises key componentsof the cell cycle regulatory machinery in defined combinations toprovide novel means of determining cell cycle status for individualliving cells in a non-destructive process providing dynamic read out.

The present invention further provides proteins, DNA constructs,vectors, and stable cell lines expressing such proteins, that exhibittranslocation of a detectable reporter molecule in a cell cycle phasespecific manner, by direct linkage of the reporter signal to a G1/S cellcycle phase dependent location control sequence. This greatly improvesthe precision of determination of cell cycle phase status and allowscontinuous monitoring of cell cycle progression in individual cells.Furthermore, it has been found that key control elements can be isolatedand abstracted from functional elements of the cell cycle controlmechanism to permit design of cell cycle phase reporters which aredynamically regulated and operate in concert with, but independently of,endogenous cell cycle control components, and hence provide means formonitoring cell cycle position without influencing or interfering withthe natural progression of the cell cycle.

According to a first aspect of the present invention, there is provideda polypeptide construct comprising a detectable live-cell reportermolecule linked via a group having a molecular mass of less than 112,000Daltons to at least one cell cycle phase-dependent location controlelement, the location of which said element changes during G1 and Sphase, wherein the translocation of said construct within a mammaliancell is indicative of the cell cycle position.

It will be understood that translocation is defined as the detectablemovement of the reporter from one sub-cellular location to another,typically from the nucleus to the cytoplasm or vice versa. It will befurther understood that the term ‘live cell’, as it relates to areporter molecule, defines a reporter molecule which produces adetectable signal in living cells, or a reporter, such as an antigenictag, that is expressed in living cells and can be detected afterfixation through immunological methods, and is thus suitable for use inimaging systems, such as the IN Cell Analyzer (GE Healthcare).

Suitably, said group has a molecular mass of less than 100,000 Daltons.

Suitably, the group has a molecular mass of less than 50,000 Daltons.

Suitably, the group has a molecular mass of less than 25,000 Daltons.

Suitably, the group has a molecular mass of less than 10,000 Daltons.

Suitably, the group has a molecular mass of less than 1,000 Daltons.

Suitably, the group has a molecular mass of less than 700 Daltons.

Suitably, the group has a molecular mass of less than 500 Daltons.

Preferably, the group is a polypeptide. The polypeptide group should berelatively small and comprise amino acids that allow flexibility and/orrotation of the reporter molecule relative to the cell cyclephase-dependent location control element. More preferably, thepolypeptide group is a heptapeptide. Most preferably, said heptapeptidegroup is Gycine-Asparagine-Glycine-Glycine-Asparagine-Alanine-Serine(GNGGNAS; SEQ ID NO: 18). As stated above, any amino acids which allowflexibility and/or rotation of the reporter molecule relative to thelocation control element may be used in the polypeptide. Suitably, thecell cycle phase-specific dependent location control element is selectedfrom the group of peptides consisting of Rag2, Chaf1B, Fen1, PPP1 R2,helicase B, sgk, CDC6 or motifs therein such as thephosphorylation-dependent subcellular localization domain of theC-terminal special control region of helicase B (PSLD). Helicase B isknown to cause uncontrolled DNA licensing and may be detrimental to cellsurvival when over-expressed. Therefore, preferably, the cell cyclephase-dependent location control element is thephosphorylation-dependent subcellular localization domain of theC-terminal spacial control region of helicase B (PSLD).

A human helicase B homolog has been reported and characterised ((Tanejaet al J. Biol. Chem., (2002), 277, 40853-40861); the nucleic acidsequence (NM 033647) and the corresponding protein sequence are given inSEQ ID No. 1 and SEQ ID No. 2, respectively. The report demonstratesthat helicase activity is needed during G1 to promote the G1/Stransition. Gu et al (Mol. Biol. Cell., (2004), 15, 3320-3332) haveshown that a small C-terminal region of the helicase B gene termed thephosphorylation-dependent subcellular localization domain (PSLD) isphosphorylated by Cdk2/cyclin E and contains NLS and NES sequences. Guet al (Mol. Biol. Cell., (2004), 15, 3320-3332) carried out studies oncells that had been transiently transfected with plasmid encoding anEGFP-βGal-PSLD fusion (beta-galactosidase (βGal) was included in theconstruct as an inert group to make the whole fusion protein similar insize to the complete helicase B) expressed from a CMV promoter. Cells inG1 exhibited EGFP signal predominantly in the nucleus, whilst cells inother phases of the cell cycle exhibited predominantly cytoplasmic EGFPsignal. These researchers concluded that the PSLD was directingtranslocation of the reporter from the nucleus to the cytoplasm aroundthe G1/S phase transition of the cell cycle.

Suitably, the live-cell reporter molecule is selected from the groupconsisting of fluorescent protein, enzyme and antigenic tag. Preferably,the fluorescent protein is derived from Aequoria Victoria, Renillareniformis or other members of the classes Hydrozoa and Anthozoa (Labaset al., Proc. Natl. Acad. Sci, (2002), 99, 4256-4261). More preferably,the fluorescent protein is EGFP (BD Clontech), Emerald (Tsien, Annu.Revs. Biochem., (1998), 67, 509-544) or J-Red (Evrogen). Mostpreferably, the fluorescent protein is selected from the groupconsisting of Green Fluorescent Protein (GFP), Enhanced GreenFluorescent Protein (EGFP), Emerald and J-Red.

Suitably, the reporter is an enzyme reporter such as halo-tag (Promega).

Suitably, the reporter molecule is EGFP or J-Red and the cell cyclephase-dependent location control element is PSLD.

Suitably, the reporter molecule is tandemized (i.e. present as a tandemrepeat).

A polypeptide construct comprising the amino acid sequence of SEQ ID No.5.

According to a second aspect of the present invention, there is provideda nucleic acid construct encoding any of the polypeptide constructs ashereinbefore described.

Suitably, said nucleic acid construct additionally comprises and isoperably linked to and under the control of at least one cell cycleindependent expression control element.

The term, ‘operably linked’ indicates that the elements are arranged sothat they function in concert for their intended purposes, e.g.transcription initiates in a promoter and proceeds through the DNAsequence coding for the reporter molecule of the invention.

Suitably, the expression control element controls transcription over anextended time period with limited variability in levels of transcriptionthroughout the cell cycle. Preferably, the expression control element isthe ubiquitin C or CMV I/E promoter which provide transcription over anextended period which is required for the production of stable celllines.

Preferably, the nucleic acid construct comprises a Ubiquitin C promoter,and sequences encoding PSLD and EGFP or J-Red.

Optionally, the nucleic acid construct comprises a CMV promoter, andsequences encoding PSLD and EGFP or J-Red.

In a third aspect of the present invention, there is provided a vectorcomprising any of the nucleic acid constructs as hereinbefore described.Suitably, said vector is either a viral vector or a plasmid. Suitably,said viral vector is an adenoviral vector or a lentiviral vector.

Optionally, the vector additionally contains a drug resistance gene thatis functional in eukaryotic cells, preferably a drug resistance genethat is functional in mammalian cells.

Expression vectors may also contain other nucleic acid sequences, suchas polyadenylation signals, splice donor/splice acceptor signals,intervening sequences, transcriptional enhancer sequences, translationalenhancer sequences and the like. Optionally, the drug resistance geneand reporter gene may be operably linked by an internal ribosome entrysite (IRES), (Jang et al., J. Virology, (1988), 62, 2636-2643) ratherthan the two genes being driven by separate promoters. The pIRES-neo andpIRES vectors commercially available from Clontech may be used.

In a fourth aspect of the present invention, there is provided a hostcell transfected with a nucleic acid construct as hereinbeforedescribed. The host cell into which the construct or the expressionvector containing such a construct is introduced may be any mammaliancell which is capable of expressing the construct.

The prepared DNA reporter construct may be transfected into a host cellusing techniques well known to the skilled person. These techniques mayinclude: electroporation (Tur-Kaspa et al, Mol. Cell Biol. (1986), 6,716-718), calcium phosphate based methods (eg. Graham and Van der Eb,Virology, (1973), 52, 456-467), direct microinjection, cationic lipidbased methods (eg. the use of Superfect (Qiagen) or Fugene6 (Roche) andthe use of bombardment mediated gene transfer (Jiao et al,Biotechnology, (1993), 11, 497-502). A further alternative method fortransfecting the DNA construct into cells, utilises the natural abilityof viruses to enter cells. Such methods include vectors and transfectionprotocols based on, for example, Herpes simplex virus (U.S. Pat. No.5,288,641), cytomegalovirus (Miller, Curr. Top. Microbiol. Immunol.,(1992), 158, 1), vaccinia virus (Baichwal and Sugden, 1986, in GeneTransfer, ed. R. Kucherlapati, New York, Plenum Press, p117-148), andadenovirus and adeno-associated virus (Muzyczka, Curr. Top. Microbiol.Immunol., (1992), 158, 97-129).

Examples of suitable recombinant host cells include HeLa cells, Verocells, Chinese Hamster ovary (CHO), U20S, COS, BHK, HepG2, NIH 3T3 MDCK,RIN, HEK293 and other mammalian cell lines that are grown in vitro.Preferably the host cell is a human cell. Such cell lines are availablefrom the American Tissue Culture Collection (ATCC), Bethesda, Md.,U.S.A. Cells from primary cell lines that have been established afterremoving cells from a mammal followed by culturing the cells for alimited period of time are also intended to be included in the presentinvention.

In a preferred embodiment, the cell line is a stable cell linecomprising a plurality of host cells according to the fourth aspect.

Cell lines which exhibit stable expression of a cell cycle positionreporter may also be used in establishing xenografts of engineered cellsin host animals using standard methods. (Krasagakis, K. J et al, CellPhysiol., (2001), 187(3), 386-91; Paris, S. et al, Clin. Exp.Metastasis, (1999), 17(10), 817-22). Xenografts of tumour cell linesengineered to express cell cycle position reporters will enableestablishment of model systems to study tumour cell division, stasis andmetastasis and to screen new anticancer drugs.

In a fifth aspect of the present invention, there is provided the use ofa polypeptide as hereinbefore described for determining the cell cycleposition of a mammalian cell.

Use of engineered cell lines or transgenic tissues expressing a cellcycle position reporter as allografts in a host animal will permit studyof mechanisms affecting tolerance or rejection of tissue transplants(Pye & Watt, J. Anat., (2001), 198 (Pt 2), 163-73; Brod, S. A. et al,Transplantation (2000), 69(10), 2162-6).

According to a sixth aspect of the present invention, there is provideda method for determining the cell cycle position of a mammalian cell,said method comprising:

-   a) expressing in a cell a nucleic acid construct as hereinbefore    described; and-   b) determining the cell cycle position by monitoring signals emitted    by the reporter molecule.

To perform the method for determining the cell cycle position of a cellaccording to the sixth aspect, cells transfected with the DNA reporterconstruct may be cultured under conditions and for a period of timesufficient to allow expression of the reporter molecule at a specificstage of the cell cycle. Typically, expression of the reporter moleculewill occur between 16 and 72 hours post transfection, but may varydepending on the culture conditions. If the reporter molecule is basedon a green fluorescent protein sequence the reporter may take a definedtime to fold into a conformation that is fluorescent. This time isdependent upon the primary sequence of the green fluorescent proteinderivative being used. The fluorescent reporter protein may also changecolour with time (see for example, Terskikh, Science, (2000), 290,1585-8) in which case imaging is required at specified time intervalsfollowing transfection.

If the reporter molecule produces a fluorescent signal in the method ofthe sixth aspect, either a conventional fluorescence microscope, or aconfocal based fluorescence microscope may be used to monitor theemitted signal. Using these techniques, the proportion of cellsexpressing the reporter molecule, and the location of the reporter canbe determined. In the method according to the present invention, thefluorescence of cells transformed or transfected with the DNA constructmay suitably be measured by optical means in for example; aspectrophotometer, a fluorimeter, a fluorescence microscope, a cooledcharge-coupled device (CCD) imager (such as a scanning imager or an areaimager), a fluorescence activated cell sorter, a confocal microscope ora scanning confocal device, where the spectral properties of the cellsin culture may be determined as scans of light excitation and emission.

In the embodiment of the invention wherein the nucleic acid reporterconstruct comprises a drug resistance gene, following transfection andexpression of the drug resistance gene (usually 1-2 days), cellsexpressing the modified reporter gene may be selected by growing thecells in the presence of an antibiotic for which transfected cells areresistant due to the presence of a selectable marker gene. The purposeof adding the antibiotic is to select for cells that express thereporter gene and that have, in some cases, integrated the reportergene, with its associated promoter, into the genome of the cell line.Following selection, a clonal cell line expressing the construct can beisolated using standard techniques. The clonal cell line may then begrown under standard conditions and will express reporter molecule andproduce a detectable signal at a specific point in the cell cycle.

Cells transfected with the nucleic acid reporter construct according tothe present invention may be grown in the absence and/or the presence ofa test agent to be studied and whose effect on the cell cycle of a cellis to be determined. By determining the proportion of cells expressingthe reporter molecule and the localisation of the signal within thecell, it is possible to determine the effect of a test agent on the cellcycle of the cells, for example, whether the test system arrests thecells in a particular stage of the cell cycle, or whether the effect isto speed up or slow down cell division.

Thus, according to a seventh aspect of the present invention, there isprovided a method of determining the effect of a test agent on the cellcycle position of a mammalian cell, the method comprising:

-   a) expressing in the cell in the absence and in the presence of the    test agent a nucleic acid reporter construct as hereinbefore    described; and-   b) determining the cell cycle position by monitoring signals emitted    by the reporter molecule wherein a difference between the emitted    signals measured in the absence and in the presence of the test    agent is indicative of the effect of the test agent on the cell    cycle position of the cell.

The term ‘test agent’ should be construed as a form of electromagneticradiation or as a chemical entity. Preferably, the test agent is achemical entity selected from the group consisting of drug, nucleicacid, hormone, protein and peptide. The test agent may be appliedexogenously to the cell or may be a peptide or protein that is expressedin the cell under study.

In an eighth aspect of the present invention, there is provided a methodof determining the effect of a test agent on the cell cycle position ofa mammalian cell, the method comprising:

-   a) expressing in said cell in the presence of said test agent a    nucleic acid reporter construct as hereinbefore described;-   b) determining the cell cycle position by monitoring signals emitted    by the reporter molecule, and-   c) comparing the emitted signal in the presence of the test agent    with a known value for the emitted signal in the absence of the test    agent;    wherein a difference between the emitted signal measured in the    presence of the test agent and the known value in the absence of the    test agent is indicative of the effect of the test agent on the cell    cycle position of the cell.

In a ninth aspect of the present invention, there is provided a methodof determining the effect of a test agent on the cell cycle position ofa mammalian cell, the method comprising:

-   a) providing cells containing a nucleic acid reporter construct as    hereinbefore described;-   b) culturing first and second populations of the cells respectively    in the presence and absence of a test agent and under conditions    permitting expression of the nucleic acid reporter construct; and-   c) measuring the signals emitted by the reporter molecule in the    first and second cell populations;    wherein a difference between the emitted signals measured in the    first and second cell populations is indicative of the effect of the    test agent on the cell cycle position of the cell.

According to a tenth aspect of the present invention, there is provideda method of determining the effect of the mammalian cell cycle on acellular process measurable by a first detectable reporter which isknown to vary in response to a test agent, the method comprising:

-   a) expressing in the cell in the presence of the test agent a second    nucleic acid reporter construct as hereinbefore described;-   b) determining the cell cycle position by monitoring signals emitted    by the second reporter molecule; and-   c) monitoring the signals emitted by the first detectable reporter,    wherein the relationship between cell cycle position determined by    step b) and the signal emitted by the first detectable reporter is    indicative of whether or not said cellular process is cell cycle    dependent.

In an eleventh aspect of the present invention, there is provided theuse of a polypeptide as hereinbefore described for measuring CDK2activity in a cell.

According to a twelfth aspect of the present invention, there isprovided a method for measuring CDK2 activity in a cell, said methodcomprising the steps of

-   a) expressing a nucleic acid construct in a cell as hereinbefore    described' and-   b) determining CDK2 activity by monitoring signals emitted by the    reporter molecule.

According to a thirteenth aspect of the present invention, there isprovided a method for determining the effect of a test agent on CDK2activity of a mammalian cell, said method comprising:

-   a) expressing in said cell in the absence and in the presence of    said test agent a nucleic acid construct as hereinbefore described;    and-   b) determining CDK2 activity by monitoring signals emitted by the    reporter molecule wherein a difference between the emitted signals    measured in the absence and in the presence of said test agent is    indicative of the effect of the test agent on the activity of CDK2.

In a fourteenth aspect of the present invention, there is provided amethod of determining the effect of a test agent on CDK2 activity of amammalian cell, said method comprising:

-   a) expressing in said cell in the presence of said test agent a    nucleic acid construct as hereinbefore described; and-   b) determining the cell cycle position by monitoring signals emitted    by the reporter molecule,-   c) comparing the emitted signal in the presence of the test agent    with a known value for the emitted signal in the absence of the test    agent;    wherein a difference between the emitted signal measured in the    presence of the test agent and said known value in the absence of    the test agent is indicative of the effect of the test agent on the    CDK2 activity of the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further illustrated by reference to the followingexamples and figures in which:

FIG. 1—Localisation of HDHB in the nucleus or cytoplasm.

(A) Cytoplasmic and nuclear extracts of U2OS cells were analyzed bydenaturing gel electrophoresis and western blotting with antibodyagainst recombinant HDHB, α-tubulin, and PCNA. Immunoreactive proteinswere detected by chemiluminescence.

(B) GFP-tagged HDHB microinjected and transiently expressed in U2OScells were visualized by fluorescence microscopy. Nuclei were stainedwith Hoechst dye. Bar, 10 μm.

(C) FLAG-tagged HDHB microinjected and transiently expressed in U2OScells were visualized by fluorescence microscopy.

FIG. 2—The subcellular localization of GFP-HDHB is cell cycle-dependent.

(A) Subcellular localization of transiently expressed GFP-tagged HDHB inasynchronous, G1, and S phase U2OS cells was quantified. The number ofGFP-positive cells with a given distribution pattern was expressed as apercentage of the total number of GFP-positive cells (>100 cells).(B) Cytoplasmic and nuclear extracts of synchronized U2OS cells (G1 andS phase) were analyzed by denaturing gel electrophoresis and westernblotting with antibody against recombinant HDHB, α-tubulin, and PCNA.Immunoreactive proteins were detected by chemiluminescence.

FIG. 3—Identification of a domain required for nuclear localization ofHDHB.

(A) Schematic representation of the HDHB protein showing seven potentialphosphorylation sites for CDK (SP or TP), the putative subcellularlocalization domain (SLD) and phosphorylated SLD (PSLD), the Walker Aand Walker B helicase motifs. Amino acid residue numbers are indicatedbelow protein.(B) GFP- and FLAG-tagged HDHB and C-terminal truncation mutantsgenerated in study. The C terminus of HDHB SLD (residues 1040-1087) andPSLD (residues 957-1087) was fused to a GFP-βGal reporter to createGFP-βGal-SLD and GFP-βGal-PSLD respectively.(C) The subcellular localization of transiently expressed GFP-HDHB-ASLDin asynchronous, G1, and S phase U2OS cells was quantified and expressedas a percentage of the total number of GFP-positive cells.

FIG. 4—GFP-βGal-PSLD subcellular localization pattern varies with thecell cycle.

(A) The subcellular localization of transiently expressed GFP-βGal,GFP-βGal-SLD, and GFP-βGal-PSLD in asynchronous, G1, and S phase U2OScells was quantified and expressed as a percentage of the total numberof GFP-positive cells.

FIG. 5—Identification of a functional rev-type nuclear export signal(NES) in SLD of HDHB.

(A) Alignment of the putative NES in HDHB with those identified in othercell cycle-related proteins (Henderson and Eleftheriou, 2000; Fabbro andHenderson, 2003). Superscripts above the amino acid sequence indicateresidue numbers. Thick arrows point to the conserved aliphatic residuesin the NES. Two pairs of residues in the putative NES in HDHB weremutated to alanine as indicated by the thin arrows to create Mut1 andMut2. HIV Rev: SEQ ID NO: 7; hBRCA1: SEQ ID NO: 8; IkBα: SEQ ID NO: 9;MAPKK: SEQ ID NO: 10; PKI: SEQ ID NO: 11; RanBP1: SEQ ID NO: 12; p53cNES: SEQ ID NO: 13; 14-3-3: SEQ ID NO: 14; hdm2: SEQ ID NO: 15; MDHB:SEQ ID NO: 16; HDHB: SEQ ID NO: 17.(B) GFP- and FLAG-tagged HDHB were transiently expressed inasynchronously growing U2OS cells with (+) or without (−) LMB to inhibitCRM1-mediated nuclear export. The subcellular localization of GFP-HDHBand FLAG-HDHB in asynchronous, G1, and S phase cells was quantified andexpressed as a percentage of the total number of GFP-positive cells inthat sample.(C) The subcellular localization of wild type and mutant GFP-HDHB andGFP-βGal-PSLD in asynchronous U2OS cells was quantified and expressed asa percentage of the total number of GFP-positive cells in that sample.

FIG. 6—Cell cycle-dependent phosphorylation of FLAG-HDHB in vivo.

(A) U2OS cells transiently expressing FLAG-HDHB (lane 1) and itstruncation mutants 1-1039 (lane 2) and 1-874 (lane 3) were labeled with[³²P] orthophosphate. Cell extracts were immunoprecipitated withanti-FLAG resin. The precipitated proteins were separated by 7.5%SDS-PAGE, transferred to a PVDF membrane, and detected byautoradiography (top) or western blotting (bottom). The positions ofmarker proteins of known molecular mass are indicated at the left.(B) FLAG-HDHB expressed in U2OS cells was immunoprecipitated withanti-FLAG resin, incubated with (+) or without (−) A-phosphatase(A-PPase) in the presence (+) or absence (−) of phosphatase inhibitors,as indicated, and analyzed by SDS-PAGE and immunoblotting with anti-HDHBantibody.(C) U2OS cells expressing FLAG-HDHB were arrested at G1/S (top) or atG2/M(bottom), and then released from the block. FLAG-HDHB was harvestedat the indicated time points, immunoprecipitated with anti-FLAG resin,treated with (+) or without (−) A-PPase, and analyzed as in (B).

FIG. 7—Identification of S967 as a major in vivo phosphorylation site inHDHB.

(A) Phosphoamino acid markers (left) and phosphoamino acids from in vivo32P-labeled FLAG-HDHB (right) were separated in two dimensions andvisualized by autoradiography. Some incompletely hydrolyzedphosphopeptides remained near the origin (+).

(B) Wild type and mutant FLAG-HDHB proteins were radiolabeled withorthophosphate in vivo, immunoprecipitated, separated by SDS-PAGE, andanalyzed by autoradiography (top) and immunoblotting with anti-HDHB(bottom).

(C) Tryptic phosphopeptides of 32P-labeled wild type and S967A mutantFLAG-HDHB were separated in two dimensions and visualized byautoradiography.

FIG. 8—Identification of cyclin E/CDK2 as the potential G1/S kinase ofHDHB S967.

(A) Tryptic phosphopeptides from FLAG-HDHB phosphorylated in vivo as inFIG. 7C, or recombinant HDHB phosphorylated in vitro by purified cyclinE/CDK2 or cyclin A/CDK2, were separated in two dimensions, eitherindividually or as a mixture, and visualized by autoradiography.(B) Proteins that co-immunoprecipitated with FLAG vector (lanes 1, 4) orFLAG-HDHB (lanes 2, 5) expressed in U2OS cells were analyzed byimmunoblotting with antibodies against HDHB (lanes 1-6), cyclin E (lanes1-3), or cyclin A (lanes 4-6). One tenth of the cell lysate used forimmunoprecipitation was analyzed in parallel as a positive control(lanes 3, 6).

FIG. 9—The subcellular localization of HDHB is regulated byphosphorylation of S967.

(A) Subcellular localization of GFP-HDHB S967A and S967D expressed inasynchronous, G1, and S phase U2OS cells was quantified.

FIG. 10—Localisation of EGFP-PSLD in asynchronous U2OS cells exhibitingstable expression of the pCORON1002-EGFP-C1-PSLD vector is cell cycledependent. Fluorescence microscopy of the same partial field of cells inwhich (A) nuclei were stained with Hoechst dye, (B) EGFP-PSLD wasvisualised, (C) nuclei were exposed to BrdU for 1 hour exposure prior tofixation and detection with Cy-5 labelled antibody to indicate cells inS-phase. (D) A graph of nuclear fluorescent intensity in both the red(Cy-5 immunofluorescent detection of BrdU) and green (EGFP-PSLD) forindividual cells present in a full field of view.

FIG. 11—Vector map of pCORON1002-EGFP-C1-PSLD.

FIG. 12—Vector map of pCORON1002-EGFP-C1-βGal-PSLD

FIG. 13—Flow cytometry data comparing brightness and homogeneity ofsignal for representative stable cell lines developed withpCORON1002-EGFP-C1-PSLD, pCORON1002-EGFP-C1-βGal-PSLD and the parentalU2OS cell line.

DETAILED DESCRIPTION OF THE INVENTION Methods

Plasmids

PGFP-HDHB and mutant derivatives (see FIGS. 4 and 6) were created byinserting full-length HDHB cDNA as a BglII/NotI fragment (Taneja et al.,J. Biol. Chem., (2002) 277, 40853-40861) into the NotI site of thepEGFP-C1 vector (Clontech, Palo Alto, Calif.). PFLAG-HDHB wasconstructed by inserting a HindIII/NotI fragment containing full-lengthHDHB cDNA into the NotI site of pFlag-CMV2 vector (Eastman Kodak Co.,Rochester, N.Y.). Tagged HDHB-SLD (1-1039) was constructed by cleavingthe tagged HDHB plasmid with NruI following the coding sequence forresidue 1034 and with NotI in the polylinker and replacing the smallfragment by a duplex adaptor oligonucleotide with a blunt end encodingresidues 1035 to 1039, a stop codon, and an overhanging NotI-compatible5′ end. To create PFLAG-HDHB (1-874), StuI-digested PFLAG-HDHB DNA wastreated with Klenow polymerase to generate blunt ends and ligated intothe pFLAG-CMV2 vector. To generate pEGFP-βGal, a DNA fragment encodingE. coli β-galactosidase (βGal) was amplified by PCR from pβGal-control(Clontech) and inserted at the 3′ end of the GFP coding sequence inpEGFP-C1, using the HindIII site. The HDHB sequence for amino acidresidues 1040-1087(SLD) and 957-1087(PSLD) were PCR amplified andinserted at the 3′ end of the βGal cDNA in pEGFP-βGal to createpGFP-βGal-SLD and PGFP-βGal-PSLD respectively. The NES mutants andphosphorylation site mutants were created in the HDHB cDNA bysite-directed mutagenesis (QuikChange, Stratagene, La Jolla, Calif.).

pCORON1002-EGFP-C1-PSLD was constructed by PCR amplification of the 390bp PSLD region from the DNA construct pGFP-Cl-βGal-PSLD. Introduction of5′ NheI and 3′ SalI restriction enzyme sites to the PSLD fragmentallowed sub-cloning into the vector pCORON1002-EGFP-C1 (GE Healthcare,Amersham, UK). The resulting 6704 bp DNA constructpCORON1002-EGFP-C1-PSLD, contains an ubiquitin C promoter, a bacterialampicillin resistance gene and a mammalian neomycin resistance gene(FIG. 11). The nucleic acid sequence of the vector is shown in SEQ IDNo. 3. Three further versions of this vector were created using standardcloning techniques (Sambrook, J. et al (1989)); the EGFP gene was firstreplaced with J-Red (Evrogen), the neomycin resistance gene was replacedwith hygromycin resistance gene and the ubiquitin C promoter wasreplaced with the CMV I/E promoter.

pCORON1002-EGFP-C1-βGal-PSLD was constructed by NheI and XmaIrestriction enzyme digest of pEGFP-Cl-βGal-PSLD and insertion of the4242 bp EGFP-βGal-PSLD fragment into pCORON1002 vector (GE Healthcare).The resulting 9937 bp DNA construct PCORON 1002-EGFP-C1-βGal-PSLD (FIG.12) contains an ubiquitin C promoter, a bacterial ampicillin resistancegene and a mammalian neomycin resistance gene. The nucleic acid sequenceof the vector is shown in SEQ ID No. 4.

The protein and nucleic acid sequence for the EGFP-PSLD fusion proteinare shown in SEQ ID No. 5 and 6, respectively.

The correct DNA sequence of all constructs and substitution mutationswas confirmed by DNA sequencing.

Antibodies

Anti-HDHB antibody was generated against purified recombinant HDHB(Bethyl Laboratories, Montgomery, Tex.) and affinity-purified onimmobilized HDHB (Harlow & Lane, Antibodies: A laboratory manual. ColdSpring Harbor Laboratory).

Cell Culture, Synchronization, Microinjection, Electroporation,Transfection and Stable Cell Line Generation

U2OS cells were cultured as exponentially growing monolayers inDulbecco-modified Eagle medium (DMEM) (Gibco BRL Lifetechnologies,Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS)(Atlanta Biologicals, Norcross, Ga.) at 37° C. Exponentially growingU2OS cells were arrested at G1/S by incubation in DMEM containing 5 mMthymidine (Sigma-Aldrich, St. Louis, Mo.), for 24 h. To release thecells into S phase, the medium was aspirated and the cells washed threetimes with warm DMEM plus 10% FBS, and incubated in fresh DMEM plus 10%FBS. Exponentially growing U2OS cells were arrested in G2/M for 16 h inDMEM containing 30 ng/ml nocodazole (Sigma-Aldrich). To release cellsinto G1, mitotic cells were collected by gently shaking them off, washedthree times with DMEM plus 10% FBS, and then plated on glass coverslipsfor microinjection, or in culture dishes for further manipulation.

Cell cycle synchronization was verified by flow cytometry as describedpreviously (Taneja et al., J. Biol. Chem., (2002) 277, 40853-40861). Inexperiments to block nuclear protein export, cells were cultured for 3 hin DMEM containing 10 ng/ml of leptomycin B (LMB) and 10 μMcycloheximide (Calbiochem, San Diego, Calif.) to prevent new proteinsynthesis. Cells plated on glass coverslips were microinjected asdescribed (Herbig et al., 1999) except that plasmid DNA rather thanprotein was injected.

For electroporation, asynchronously growing U2OS cells (5×106) weretrypsinized, collected by centrifugation, and resuspended in 800 μl of20 mM HEPES (pH 7.4), 0.7 mM Na2HPO4/NaH2PO4, 137 mM NaCl, 5 mM KCl, 6mM glucose at a final pH of 7.4. Ten μg of DNA was added, transferred toa 0.4 cm electroporation cuvette (BioRad, Hercules, Calif.) andelectroporation performed using Gene Pulser II apparatus (BioRad). Cellswere plated in tissue culture dishes for 1 h, washed with fresh mediumand cultured for another 23 h.

Working with transiently transfected cells proved difficult in multiwellplate format due to low transfection efficiency, heterogeneity ofexpression and problems arising from the high throughput analysis ofsuch data. Screening for the effects of large numbers of siRNA or agentsupon the cell cycle therefore required production of a homogenous stablecell line. Due to the toxic effects of HDHB when overexpressed for longperiods a stable cell line was generated with the PSLD region linked toa reporter. U-2OS cells were transiently transfected withPCORON1002-EGFP-C1-PSLD (FIG. 11), PCORON1002-EGFP-C1-βGal-PSLD (FIG.12) or J-Red derivatives of the above vectors. Stable clones expressingthe recombinant fusion proteins were selected using 1 mg/ml G418 (Sigma)or hygromycin, where appropriate. Isolated primary clones (˜60 perconstruct) were analysed by flow cytometry to confirm the level andhomogeneity of expression of the sensor and where appropriate secondaryclones were developed using methods above.

Fluorescence Microscopy

For indirect immunofluorescence staining, cells were washed three timeswith phosphate buffered saline (PBS), fixed with 3.7% formaldehyde inPBS for 20 min, permeabilized for 5 min in 0.2% Triton X-100, andincubated with 10% FBS in PBS for 45 min. FLAG-HDHB was detected withmouse monoclonal anti-FLAG antibody (Sigma-Aldrich), 1:100 in PBS plus10% FBS for 2 h at room temperature. After washing, cells were incubatedwith Texas Red-conjugated goat anti-mouse secondary antibody (JacksonImmunoResearch Laboratories, West Grove, Pa.) at 1:100 in PBS plus 10%FBS for 1 h at room temperature. After three washes, cells wereincubated for 10 min with Hoechst 33258 (2 μM in PBS). Coverslips weremounted in ProLong Antifade (Molecular Probes, Eugene, Oreg.). Imageswere obtained with a Hamamatsu digital camera using the Openlab 3.0software (Improvision, Lexington, Mass.) on the Zeiss Axioplan 2 Imagingsystem (Carl Zeiss Inc.). The number of cells that exhibited eachpattern of subcellular localization was counted and expressed as apercentage of the total number of cells scored (100 to 150 cells in eachexperiment). The subcellular distribution of each protein wasquantitatively evaluated in at least two independent experiments.

For GFP fluorescence, cells were washed three times withphosphate-buffered saline (PBS), fixed with 3.7% formaldehyde containing2 μM Hoechst 33258 for 20 min and imaged and evaluated as above.

For Triton X-100 extraction, cells were washed twice with coldcytoskeleton buffer (CSK, 10 mM HEPES [pH 7.4], 300 mM sucrose, 100 mMNaCl, 3 mM MgCl2), and extracted for 5 min on ice with 0.5% Triton X-100in CSK buffer (supplemented with 1× protease inhibitors) and then fixedas described above.

Where appropriate, for high throughput imaging, kinetic imaging (24 hr)and analysis in multiwell plate format of stable cell lines flourescencemicroscopy was conducted using a high throughput confocal imaging system(IN Cell Analyzer 1000 or IN Cell Analyzer 3000, GE Healthcare,Amersham, UK) on cells transfected with pCORON1002-EGFP-C1-PSLD,pCORON1002-EGFP-C1-βGal-PSLD or redFP derivatives of these vectors.Images were analysed using the cell cycle phase marker algorithm (GEHealth Care).

Metabolic Phosphate Labeling

U2OS cells (2.5×106) were transiently transfected with wild type ormutant FLAGHDHB. After 24 h, cells were incubated in phosphate-depletedDMEM (Gibco BRL Lifetechnologies) for 15 min and radiolabeled with32P-H3PO4 (0.35 mCi/ml of medium; ICN Pharmaceuticals Inc., Costa Mesa,Calif.) for 4 h. Phosphate-labeled FLAG-HDHB was immunoprecipitated fromextracts, separated by 7.5% SDS/PAGE, and transferred to apolyvinylidene difluoride (PVDF) membrane as described below.

Cell Extracts, Immunoprecipitation, and Western Blotting

At 24 h after transfection, FLAG-HDHB-transfected cultures to beanalyzed by immunoprecipitation and immunoblotting were lysed in lysisbuffer (50 mM Tris-HCl pH 7.5, 10% glycerol, 0.1% NP-40, 1 mM DTT, 25 mMNaF, 100 μg/ml PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin) (0.5 ml per35 mm or 1 ml per 60 mm dish or 75 cm flask). The extract was scrapedoff the dish, incubated for 5 min on ice, and centrifuged for 10 min at14 000 g. Samples of the supernatant (0.5 to 1 mg of protein) wereincubated with 10 μl anti-FLAG agarose (Sigma) on a rotator for 2 h at4° C. The agarose beads were washed three times with lysis buffer.Immunoprecipitated proteins were transferred to a PVDF membrane andanalyzed by western blotting with anti-HDHB-peptide serum (1:5000),anti-cyclin E antibody (1:1000), and anticyclin A antibody (1:1000)(Santa Cruz Biotechnology Inc., Santa Cruz, Calif.), andchemiluminescence (SuperSignal, Pierce Biotechnology Inc., Rockford,Ill.).

For selective nuclear and cytoplasmic protein extraction, 80-90%confluent U2OS cells were harvested by trypsinization and washed withPBS. They were resuspended and lysed in 10 mM Tris-HCl [pH 7.5], 10 mMKCl, 1.5 mM MgCl2, 0.25 M sucrose, 10% glycerol, 75 μg/ml digitonin, 1mM DTT, 10 mM NaF, 1 mM Na3VO4, 100 μg/ml PMSF, 1 μg/ml aprotinin, and 1μg/ml leupeptin for 10 min on ice, and centrifuged at 1000×g for 5 min.The supernatant fraction was collected as the cytosolic extract. Thepellet was washed, resuspended in high salt buffer (10 mM Tris-HCl [pH7.5], 400 mM NaCl 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1% NP-40, 100 μg/mlPMSF, 1 μg/ml aprotinin, and 1 μg/ml leupeptin), and rocked for 10 minat 4° C. After sonication, the suspended material, containing bothsoluble and chromatin-bound protein, was analyzed as nuclear extract.Proteins in the nuclear and cytoplasmic extracts were analyzed by 8.5%SDS-PAGE, followed by western blotting with antibodies againstα-tubulin, PCNA (both Santa Cruz Biotechnology), and recombinant HDHB.

Protein Phosphatase Reactions

FLAG-HDHB bound to anti-FLAG beads was incubated with 100 U ofλ-phosphatase (New England Biolabs, Beverly, Mass.) in phosphatasebuffer (50 mM Tris-HCl [pH 7.5], 0.1 mM EDTA, 0.01% NP-40) for 1 h at30° C. The reaction was carried out in the presence or absence ofphosphatase inhibitors (5 mM Na3VO4, 50 mM NaF). The proteins wereseparated by 7.5% SDSPAGE (acrylamide-bisacrylamide ratio, 30:0.36) andHDHB was detected by western blotting with anti-HDHB-peptide serum andchemiluminescence.

Tryptic Peptide Mapping and Phosphoamino Acid Analysis

At 24 h after transfection, radiolabeled FLAG-HDHB-transfected culturesto be used for immunoprecipitation and phosphoamino acid orphosphopeptide mapping were processed as above, except that lysis bufferwas substituted by RIPA buffer (50 mM Tris-HCl [pH7.5], 150 mM NaCl, 1%NP-40, 0.5% deoxycholic acid, 1% SDS, 50 mM NaF, 1 mM EDTA, 5 mM Na3VO4,100 μg/ml PMSF, 1 μg/ml aprotinin, and 1 μg/ml leupeptin).Immunoprecipitated proteins were separated by 7.5% SDS-PAGE andtransferred to PVDF membranes. The membranes containing radiolabeledHDHB were rinsed well with deionized H₂O twice before visualization ofphosphoproteins by autoradiography. The phosphoproteins were thenexcised, and the membrane pieces were re-wet with methanol followed bywater. The membranes were blocked with 50 mM NH4HCO3 containing 0.1%Tween 20 (Sigma-Aldrich) for 30 min at room temperature and washed threetimes with 50 mM NH4HCO3 before enzymatic cleavage of phosphoproteinsfrom the PVDF with L-(tosylamido-2-phenyl) ethyl chloromethylketonetreated bovine pancreatic trypsin (Worthington, Lakewood, N.J.).The peptides were then subjected to two-dimensional phosphopeptidemapping or phosphoamino acid analysis as described in detail elsewhere(Boyle et al., Meth. Enzymology, (1991), 201, 110-149).

Cyclin-Dependent Kinase Reactions In Vitro

Kinase reactions using purified cyclin/CDK (200 pmol/h) (provided by R.Ott and C. Voitenleitner) and purified recombinant HDHB (Taneja et al.,J. Biol. Chem., (2002) 277, 40853-40861) as the substrate were performedas described previously (Voitenleitner et al., Mol. Cell. Biol., (1999),19, 646-56).

BrdU Labelling, Identification of Chemical Cell Cycle Blocks and RNAiExperiments on Stable Cell Lines

Stable cells expressing the pCORON1002-EGFP-C1-PSLD construct, wereseeded at 0.3×105/ml in 96-well Greiner plates using antibiotic-freemedium (100 μl/well) and incubate for 16 hours.

To demonstrate the distribution of EGFP-PSLD in S-phase, stable cellswere marked with BrdU for 1 hr using the cell proliferation kit(Amersham Biosciences, GE Health Care). Cells were fixed in 2% formalinand incorporated BrdU was detected by immunofluorescence with a Cy-5labelled secondary antibody system (Cell proliferation kit; GE HealthCare). Nulcei were stained with hoechst (2 μM).

For chemical block studies (Table 1), stable cells were exposed toolomoucine, roscovitine, nocodazole, mimosine, colcemid or colchicine(Sigma). Cells were fixed in 2% formalin and nuclei stained with hoechst(2 μM).

For siRNA studies, siRNA pools (Dharmacon) against certain cyclins, MCMproteins, CDKs, polo-like kinase (PLK), and a random control duplex(Table 2) were diluted in lipofectamine/optimem I (Invitrogen) to 25 nMand added to stable cells for 4 hrs. The medium was replaced and platesincubated for 48 hr. Cells were fixed in 2% formalin and nuclei stainedwith hoechst (2 μM).

After high throughput imaging and analysis on the IN Cell Analyzersystem (GEHC), data for average nuclear intensity and N:C ratio (EGFPsignal), nuclear size (hoescht signal) and, where appropriate, nuclearsignal intensity (BrdU) were obtained for the total number of individualcells in a field of view using hoescht as a nuclear mask and the IN CellAnalyzer 3000 cell cycle phase marker algorithm (GEHC). For each well,the total number of cells per field of view were catagorised intoG1-phase (predominantly nuclear EGFP distribution; high EGFP-PSLDnuclear intensity and N:C ratio), S-phase (nuclear BrdU signal >3SDsabove background; EGFP-PSLD N:C ratio around 1) and G2-phase (largenuclear size; low EGFP-PSLD N:C ratio). Although it was possible todifferentiate M-phase cells (based on small nuclear size and veryintense EGFP signal) very few such cells were seen in wells fixed withformalin since they were removed during the washing and fixationprocess.

Results

HDHB Resides in Nuclear Foci or in the Cytoplasm

To determine the subcellular localization of endogenous HDHB, nuclearand cytoplasmic proteins were selectively extracted from human U2OScells, separated by denaturing gel electrophoresis, and analyzed bywestern blotting (FIG. 1). The presence of PCNA and α-tubulin in eachextract was first monitored to assess the extraction procedure. PCNA wasenriched in the nuclear extract and not in the cytoplasmic fraction,while α-tubulin was found primarily in the cytoplasmic fraction,validating the fractionation. HDHB was detected in both the nuclear andcytoplasmic fractions (FIG. 1). The cytoplasmic HDHB migrated moreslowly than the nuclear fraction (FIG. 1), suggesting the possibility ofpost-translational modification.

These results could indicate either that HDHB was distributed throughoutthe cell, or that a mixed population of cells contained HDHB in eitherthe nucleus or the cytoplasm. To distinguish between these alternatives,HDHB was localized in situ in single cells; GFP- and FLAG-tagged HDHBwere expressed in human U2OS cells by transient transfection. Sinceprolonged over-expression of tagged or untagged HDHB was cytotoxic, allexperiments were conducted in the shortest time period possible (usually24 h). Tagged HDHB localization was analyzed in individual cells byfluorescence microscopy. Both GFP-HDHB and FLAG-HDHB displayed two majorpatterns of localization, either in the nucleus in discrete foci or inthe cytoplasm (FIG. 1). GFP-HDHB transiently expressed in primary humanfibroblasts was also observed in either the nucleus or the cytoplasm.

Identification of a Cell Cycle-Dependent Subcellular Localization Domainin HDHB

U2OS cells were arrested in G2/M with nocodazole, released into G1 forthree hours, and then microinjected with PGFP-HDHB DNA into theirnuclei. GFP-HDHB expression was easily detectable six hours later, whenapproximately 70% of G1 phase cells had accumulated the fusion proteinprimarily in the nuclei (FIG. 2). In contrast, when cells weresynchronized at G1/S with thymidine, released into S phase, and thenmicroinjected with PGFP-HDHB DNA, more than 70% of S phase cells hadaccumulated the fusion protein predominantly in the cytoplasm (FIG. 2).Selective extraction of U2OS cells in G1 and S phase revealed thatendogenous HDHB was mostly nuclear in G1 and cytoplasmic in S phase(FIG. 2 b). However, endogenous HDHB was clearly detectable in bothsubcellular fractions. The mobility of the S phase HDHB was slightlyretarded compared to the G1 phase protein. These results indicate thatthe subcellular localization of HDHB is regulated in the cell cycle andthat GFP-tagged HDHB reflects the localization of the endogenousuntagged helicase.

Prompted by the identification of C-terminal nuclear location signals inBloom's syndrome helicase and other RecQ-family helicases (Hickson,Nature Rev. Cancer, (2003) 3, 169-178), a possible subcellularlocalization domain (SLD) was identified at the extreme C-terminus ofHDHB (FIG. 3). To determine whether this putative SLD was important forHDHB localization, a truncation mutant of HDHB (GFP-HDHB-.SLD) wasgenerated that lacks the C-terminal 48 residues containing the SLD (FIG.3). The expression vector was microinjected into U2OS cells in G1 or Sphase and the subcellular localization of the fusion protein wasexamined by fluorescence microscopy six hours later. Over 95% of thecells accumulated the fusion protein in the cytoplasm, regardless of thecell cycle timing of HDHB expression (FIG. 3 c). This result suggeststhat HDHB may carry a NLS that is impaired or abolished by theC-terminal deletion in GFP-HDHB-ΔSLD.

To determine whether the C-terminal domain of HDHB was sufficient fornuclear localization, a bacterial β-galactosidase (βGal) was used as areporter protein because it has a molecular mass (112 kDa) close to thatof HDHB and does not contain subcellular localization signals (Kalderonet al., Cell, (1984), 39, 499-509). As a control, a GFP-βGal expressionvector (FIG. 3) was created and the subcellular localization of thefusion protein monitored after microinjection of the expression vectorinto U2OS cells. As expected, GFP-βGal protein accumulated primarily inthe cytoplasm (FIG. 4). In contrast, GFP-βGal-SLD was found in both thenucleus and cytoplasm in asynchronous or synchronized U2OS cells (FIG.4), suggesting that SLD contains a NLS, but was not sufficient fornuclear localization of the reporter protein. Reasoning that perhaps theneighboring potential CDK phosphorylation sites might affect subcellularlocalization in the cell cycle (FIG. 3), a GFP-βGal-PSLD wasconstructed, in which the C-terminal 131 residues of HDHB, containingthe putative SLD and the cluster of potential CDK phosphorylation sites,were appended to the C-terminus of GFP-βGal (FIG. 3). When theGFP-βGal-PSLD plasmid DNA was transiently expressed in asynchronous andsynchronized U2OS cells, GFP-βGal-PSLD was found in the nucleus in over90% of G1 phase cells, and in the cytoplasm in more than 70% of S phasecells (FIG. 4). In contrast with the focal pattern observed for nuclearGFP-HDHB in G1, GFP-βGal-PSLD and EGFP-PSLD proteins were distributedevenly throughout the nucleus in G1, sparing only the nucleoli. Analysisof stable cell lines expressing pCORON1002-EGFP-C1-PSLD that have beenmarked with BrdU emphasized that cells in S-phase (equal to approx 60%of the asychronous population) exhibit equidistribution or predominantlycytoplasmic distribution of the EGFP-PSLD signal (FIG. 10). S-phasecells do not show a predominantly nuclear distribution of EGFP-PSLDassociated with G1 cells. Some cells were seen to exhibit absolutenuclear exclusion of the EGFP-PSLD reporter (FIG. 10) however thesecells did not incorporate BrdU. We hypothesised that cells demonstratingabsolute clearance of EGFP-PSLD from the nucleus were in G2. Kineticimaging of the EGFP-PSLD stable cell lines over 24 hours showed thatEGFP-PSLD is predominantly nuclear in G1 after mitosis, exhibits a rapidnuclear to cytoplasmic movement around the G1/S transition (˜3.5 hoursafter cytokinesis) and further progressive translocation from thenucleus to the cytoplasm from G1/S through to the end of G2 (approx 19hours); at this point cell rounding occurred prior to re-division. Theseobservations seem to confirm the possibility that G2 cells exhibit anabsolute cytoplasmic distribution of the EGFP-PSLD reporter. Stableexpression of the EGFP-PSLD fusion was not found to affect the totallength of the cell cycle (approx 24 hours) when compared to U2OS cellsor the G2M cell cycle phase marker cell line (GEHC). Taken together,these data suggest that the subcellular localization of HDHB isdependent on the cell cycle, that the C-terminal PSLD domain of HDHBplays a major role in regulating the subcellular localization of theprotein in a cell cycle dependent manner and that HDHB is nuclear in G1but progressively translocates to the cytoplasm during S-phase andpossibly G2.

Identification of a Functional Rev-Type NES in HDHB

A number of proteins that shuttle between the nucleus and cytoplasm havebeen demonstrated to contain a NES similar to the prototype NES of HIVrev protein (FIG. 5). Proteins containing a rev-type NES require theexport factor CRM1 (also called exportin 1) to bind and transportproteins from the nucleus to the cytoplasm (reviewed by Weis, Cell,(2003), 112, 441-451). Leptomycin B (LMB), specifically inhibits CRM1activity in nuclear protein export (Wolff et al., Chem. Biol., (1997),4, 139-147; Kudo et al., Exp. Cell. Res., (1998), 242, 540-547).Inspection of the PSLD sequence in HDHB revealed a putative rev-type NES(LxxxLxxLxL; FIG. 5). To determine whether the cytoplasmic localizationof HDHB requires a functional NES, expression plasmids for GFP-HDHB orFLAG-HDHB DNA were microinjected into asynchronous, G1, and S phasecells in the presence and absence of LMB. The localization of the fusionproteins was examined by fluorescence microscopy and quantified. In thepresence of LMB, both fusion proteins accumulated in the nucleusindependently of the cell cycle (FIG. 5), consistent with thepossibility that HDHB contains a rev-type NES that functions throughCRM1. However it is also possible that HDHB may not be a direct cargo ofCRM1 and that its export may be indirectly mediated through some otherprotein(s). To assess whether the putative NES in HDHB was functional,we mutated Val/Leu and Leu/Leu of the NES motif to alanine to create NESmutants 1 and 2 (FIG. 5). GFP-HDHB and GFP-βGal-PSLD harboring these NESmutations were transiently expressed in either asynchronous orsynchronized U2OS cells. Both NES mutant fusion proteins accumulated inthe nucleus in more than 80% of cells, no matter when they wereexpressed in asynchronous or synchronized cells (FIG. 5). The resultsindicate that the NES mutations specifically impaired the export of bothGFP-HDHB and GFP-βGal-PSLD, arguing that the PSLD region of HDHBcontains a functional NES.

FLAG-HDHB is Phosphorylated in a Cell Cycle-Dependent Manner In vivo.

The cluster of potential CDK phosphorylation sites in the PSLD domain ofHDHB (FIG. 3) suggested that phosphorylation of HDHB might regulate itssubcellular localization in the cell cycle. If so, one would expect thePSLD region of HDHB to be phosphorylated in a cell cycle-dependentmanner. To test whether HDHB undergoes phosphorylation in PSLD, U2OScells were transiently transfected with expression plasmids for wildtype and C-terminally truncated forms of FLAG-HDHB, radiolabeled withphosphate, and then FLAG-HDHB was immunoprecipitated from cell extracts.Immunoprecipitated proteins were analyzed by denaturing gelelectrophoresis, immunoblotting, and autoradiography (FIG. 6). Aradiolabeled band of FLAG-HDHB was detected at the same position as theimmunoreactive HDHB band (FIG. 6A, lanes 1). Truncated FLAG-HDHB lackingSLD was also robustly phosphorylated in vivo (lanes 2), while truncatedFLAG-HDHB (1-874) lacking PSLD was not significantly phosphorylated(lanes 3). These results demonstrate that SLD is not required for HDHBphosphorylation, while PSLD is required, and suggest that thephosphorylation sites probably reside in PSLD.

To examine the timing of HDHB phosphorylation in the cell cycle, itwould be convenient to detect phosphorylation without the use ofradiolabeling. Since phosphorylation often reduces the electrophoreticmobility of a protein in denaturing gels, transiently expressedFLAG-HDHB was immunoprecipitated and its mobility examined before andafter treatment with λ-phosphatase (λ-PPase) (FIG. 6B). Without λ-PPasetreatment, FLAG-HDHB was detected in western blots in two very closelymigrating bands (lane 1), while dephosphorylated FLAG-HDHB migrated as asingle band at the mobility of the faster band of the doublet (lane 2).When λ-PPase inhibitors were present in the reaction, FLAG-HDHB migratedas a doublet identical to the mock-treated protein (lane 3). These datasuggest that the electrophoretic mobility of FLAG-HDHB was reduced byphosphorylation and that this assay may be suitable to track HDHBphosphorylation in the cell cycle.

To determine whether HDHB is phosphorylated in a cell cycle-dependentmanner, U2OS cells transiently expressing FLAG-HDHB were arrested inG1/S by adding thymidine to the medium or in G2/M by adding nocodazoleto the medium. The cells were released from the blocks for differenttime periods, and FLAG-HDHB was immunoprecipitated from cell extracts.

The immunoprecipitated material was incubated with or without λ-PPaseand then analyzed by denaturing gel electrophoresis and western blotting(FIG. 6C). The mobility of FLAG-HDHB from cells arrested at G1/S wasincreased by λ-PPase treatment, suggesting that the protein wasphosphorylated at G1/S (FIG. 6C, upper panel). A similar mobility shiftwas detected after phosphatase treatment of FLAG-HDHB for at least ninehours after release from the G1/S block (upper panel), as well as incells arrested at G2/M (FIG. 6C, lower panel). However, after the cellswere released into G1 for four and eight hours, FLAG-HDHB migrated as asingle band that was much less affected by phosphatase treatment (FIG.6C, lower panel). By twelve hours after release from the G2/M block,when most of the cells were entering S phase (data not shown), themobility of FLAG-HDHB was again increased by phosphatase treatment,restoring the pattern observed in nocodazole-arrested cells (lowerpanel). These results strongly suggest that phosphorylation of FLAG-HDHBis cell cycle-dependent, with maximal phosphorylation from G1/S throughG2/M and minimal phosphorylation during G1.

Serine 967 is the Major Phosphorylation Site of Ectopically ExpressedHDHB.

To map the phosphorylation sites in FLAG-HDHB, we first wished todetermine what amino acid residues were modified. Phosphoamino acidanalysis of in vivo radiolabeled FLAG-HDHB revealed thatphosphoserine(s) was the major phosphoamino acid of FLAG-HDHB in vivo(FIG. 7A). Assuming that the cell cycle-dependent phosphorylation sitesof HDHB are located in PSLD between residues 874 and 1039 (FIG. 3A),that these sites are modified by CDKs, and that phosphoserine is themajor amino acid modified (FIG. 7A), only four of the seven potentialCDK sites would remain as candidate sites. To test each of these sitesindividually, FLAG-HDHB expression plasmids with the correspondingserine to alanine mutations were constructed. Cells transientlytransfected with these plasmids were radiolabeled with orthophosphate invivo and FLAG-HDHB was immunoprecipitated and analyzed byautoradiography and western blotting (FIG. 7B). The results showed thatFLAG-HDHB and three of the mutant proteins were phosphorylatedapproximately equally, while the S967A mutant protein was only weaklyphosphorylated (FIG. 7B). This result suggested that S967 might be theprimary site of HDHB phosphorylation in vivo. Consistent with thisinterpretation, an electrophoretic mobility shift after phosphatasetreatment of immunoprecipitated FLAG-HDHB was detected with three of themutant proteins, but not with S967A protein.

To confirm that S967 was the major phosphorylation site in HDHB in vivo,tryptic phosphopeptide mapping was carried out with wild type and S967Amutant FLAG-HDHB that had been metabolically radiolabeled withorthophosphate (FIG. 7C). One predominant radiolabeled peptide and aweakly labeled peptide were observed with the wild type protein (leftpanel). The predominant phosphopeptide was absent in the S967A protein,but the weakly labeled peptide remained detectable (FIG. 7C, rightpanel). The results provide additional evidence that serine 967 is aprominent phosphorylation site in HDHB in vivo.

Identification of Cyclin E/CDK2 as a Kinase that Potentially ModifiesHDHB in G1/S

To test whether CDKs can actually modify HDHB, as suggested by thetiming of HDHB phosphorylation in the cell cycle and the identificationof S967 as a primary site of modification, purified cyclin E/CDK2 orcyclin A/CDK2 were incubated with purified recombinant HDHB andradiolabeled ATP in vitro. After the kinase reactions, the proteins wereseparated by denaturing gel electrophoresis, transferred to a PVDFmembrane, and detected by autoradiography. The results revealed thatrecombinant HDHB could be phosphorylated strongly by both cyclin E/CDK2and cyclin A/CDK2. The radiolabeled HDHB bands were then furtherprocessed for tryptic phosphopeptide mapping. Peptides from eachdigestion were separated in two dimensions, either individually or aftermixing with tryptic peptides from in vivo phosphorylated FLAG-HDHB, andvisualized by autoradiography (FIG. 8A). HDHB peptides phosphorylated bycyclin E/CDK2 and cyclin A/CDK2 yielded patterns essentially identicalto those observed in the in vivo labeled peptide map, with one majorspot and one minor spot (FIG. 8A). When the in vitro and in vivo labeledpeptides were mixed and separated on one chromatogram, they co-migrated(FIG. 8A, right). These data argue that the major phosphopeptidesmodified in vitro by cyclin E/CDK2 and cyclin A/CDK2 in purifiedrecombinant HDHB were the same ones modified in vivo in FLAG-HDHB.

Since cyclin E activity in human cells rises in late G1, while cyclin Aactivity rises later coincident with the onset of S phase (Pines, 1999;Erlandsson et al., 2000), it was important to try to distinguish whetherone of these kinases might preferentially modify HDHB. Cyclin subunitsfrequently form a complex with the substrate proteins that they targetfor phosphorylation (Endicott et al., 1999; Takeda et al., 2001). Totest whether cyclin E or cyclin A could associate with HDHB, FLAG-HDHBand associated proteins were immunoprecipitated from extracts of cellstransfected with either FLAG-HDHB expression vector or empty FLAG vectoras a control. The cell extracts and the immunoprecipitated material wereanalyzed by western blotting (FIG. 8B). Cyclin E clearly co-precipitatedwith FLAG-HDHB, but cyclin A did not (FIG. 8B, lanes 2 and 5),suggesting that FLAG-HDHB may interact preferentially with cyclin E invivo. It is conceivable that this interaction may be required forphosphorylation of HDHB by cyclin E/CDK2 in vivo, and if so, mutationsin HDHB that prevent its association with cyclin E would abrogatephosphorylation by cyclin E/CDK2. To test the possibility that theFLAG-HDHB mutant S967A was not phosphorylated in vivo (FIG. 7B, C) dueto an inability to bind to cyclin E, FLAG-HDHB-S967A and associatedproteins were immunoprecipitated from extracts of transfected cells andanalyzed by western blotting. Co-precipitation of cyclin E with themutant protein was as robust as with wild type FLAG-HDHB.

Phosphorylation of Serine 967 is Critical for Regulation of HDHBLocalization.

The data above indicate that subcellular localization andphosphorylation of ectopically expressed HDHB were regulated in a cellcycle-dependent manner with maximal phosphorylation from G1/S to G2/M,coinciding with the period when HDHB accumulated in the cytoplasm. Theseresults, together with the identification of S967 as the major in vivophosphorylation site in HDHB, suggest that phosphorylation of S967 mayregulate the subcellular localization of HDHB. To test this idea,expression plasmids for wild type GFP-HDHB and the mutants S967A, S984A,S1005A, and S1021A were microinjected into synchronized U2OS cells. Wildtype GFP-HDHB accumulated in nuclear foci of cells in G1, but in thecytoplasm of cells in S phase as expected. However, regardless of cellcycle timing, GFP-HDHB-S967A localized in nuclear foci in about 70% ofthe fluorescent cells (FIG. 9). The other three substitution mutantslocalized in either the nucleus or the cytoplasm like wild typeGFP-HDHB. In an attempt to mimic the phosphorylation of S967, serine 967was mutated to aspartic acid, GFP-HDHB-S967D was expressed inasynchronous and synchronized U2OS cells, and the subcellulardistribution of the mutant fusion protein was examined.

About 60% of the cells expressing GFP-HDHB-S967D displayed cytoplasmicfluorescence in asynchronous, G1 phase, and S phase cells (FIG. 9A),demonstrating that the S967D mutation mimicked phosphorylated S967. Thedata strongly suggest that phosphorylation of serine 967 is critical inregulating the subcellular localization of HDHB.

A C-Terminal Domain of HDHB Confers Cell Cycle-Dependent Localization

A 131-residue domain, PSLD, is sufficient to target HDHB, EGFP or a βGalreporter to either the nucleus or the cytoplasm in a cellcycle-dependent manner (FIGS. 4 and 10). A rev-type NES resides in thisdomain (FIG. 5), but its activity or accessibility to the nuclear exportmachinery depends on phosphorylation of PSLD, primarily on serine 967,at the G1/S transition (FIG. 6-9). S967 is a perfect match to theconsensus CDK substrate recognition motif (S/T)PX(K/R). Both cyclinE/CDK2 and cyclin A/CDK2 can modify HDHB in vitro, but the ability ofcyclin E/CDK2 to complex with HDHB in cell extracts suggests that it maybe the initial kinase that modifies HDHB at the G1/S transition (FIG.8). Addition of olomoucine and roscovitin, known Cdk2 inhibitors (Table1), or siRNA toward cyclin E (Table 2) resulted in predominantly nucleardistribution of EGFP-PSLD and arrest in G1 for EGFP-PSLD stable celllines, further supporting the possibility that Cdk2/cyclin E isresponsible for control of the observed cell-cycle basedphosphorylation-dependent subcellular localisation. Phosphorylation ofPSLD appears to persist through the latter part of the cell cycle,correlating well with the predominantly cytoplasmic localization of HDHBin S and G2. Kinetic imaging of stable cell lines treated witholomoucine over 24 hours showed that, for cells arrested in G2 theEGFP-PSLD signal redistributes from the cytoplasm to the nucleus over˜4-8 hours (without the cell passing through mitosis) suggesting that inthe absence of cdk2 activity the EGFP-PSLD either becomesdephosphorylated and re-enters the nucleus, or is destroyed and newlysynthesised protein is not phosphorylated due to cdk2 inhibition andtherefore locates in the nucleus.

TABLE 1 % Total Compound S G1 G2 cells Colcemid (0.3 μM) 41 16 43 490Colcemid (1.2 μM) 32 8 59 450 Colchicine (4 μM) 36 9 55 467 Colchicine(100 μM) 32 12 57 439 L-mimosine (2 mM) 68 6 26 1710 Olomoucine (500 μM)33 63 4 600 Roscovitin (100 μM) 36 52 13 693 Nocodazole (3 μM) 33 6 61606 Control 61 17 22 2137

TABLE 2 % Total siRNA S G1 G2 cells PLK 53 9 38 66 MCM7 58 13 29 231MCM6 64 14 22 166 MCM5 63 17 20 260 MCM4 56 20 24 223 MCM3 59 23 19 188MCM2 50 24 26 266 Cyclin B1 49 36 15 280 V2 Cyclin B1 60 24 17 203 V1CDK8 50 23 27 299 CDK7 56 18 26 354 CDK6 58 22 20 328 Cyclin A2 61 13 26319 Cyclin A1 66 10 24 298 Cyclin T2b 57 12 31 267 Cyclin T2b 55 22 23355 cyclinT1 60 20 20 260 cyclinE1 49 27 24 272 Control 69 10 20 262

It was not possible to distinguish whether HDHB undergoesdephosphorylation at the M/G1 transition (FIG. 6C) or is perhapstargeted for proteolysis and rapidly re-synthesized in early G1, when itwould enter the nucleus. However, kinetic imaging of stable cell linesover 24 hours showed that the EGFP-PSLD signal is not greatly reducedduring M phase or at the M/G1 boundary, but becomes predominantlynuclear approximately 30 minutes after cytokinesis (this state thenpersists for ˜3 hours during G1), coincident with nuclear membraneformation. This indicates that the EGFP-PSLD construct isdephosphorylated rather than undergoing significant destruction aroundthe M/G1 boundary.

These data provide strong evidence that the PSLD contains activetargeting signals that are independent of protein context (FIG. 2-5,10). Since mutant HDHB with an inactivated NES is nuclear even when itis expressed during S phase and thus presumably phosphorylated (FIG. 5),it is probable that the NLS is not inactivated by phosphorylation andthat the primary target of CDK regulation is the NES. Extending thisreasoning, the NES may be masked during G1 when the CDK motifs in PSLDare unmodified, and that the NES is liberated when S967 becomesphosphorylated, leading to NES recognition by nuclear export factors(FIG. 3-5). Structural studies of a rev-type NES have shown that itforms an amphipathic α-helix, with the leucines aligned on one side ofthe helix and charged residues on the other side (Rittinger et al., Mol.Cell. Biol. (1999), 4, 153-166). Since the SLD of HDHB contains both therev-type NES and an NLS, and the basic residues likely to serve as theNLS are interspersed through the NES, the NES and NLS may reside onopposite faces of an amphipathic helix. Additional sequences in PSLDwould mask the NES intramolecularly, allowing only the NLS to berecognized. Phosphorylation of S967 would alter the conformation of themask in PSLD to expose the NES, without affecting exposure of the NLS.

High throughput Screening for Inhibitors of the Cell Cycle withEGFP-PSLD Stable Cell Lines

As stated above, working with transiently transfected cells proveddifficult in multiwell plate format due to low transfection efficiency,heterogeneity of expression and problems arising from the highthroughput analysis of such data. Screening for the effects of largenumbers of siRNA or agents upon the cell cycle therefore requiredproduction of a homogenous stable cell line. A stable cell line wasgenerated with the PSLD region linked to a reporter (EGFP) via aflexible seven amino acid linker (using pCORON1002-EGFP-C1-PSLD). As canbe seen from FIG. 13, the fluorescent signal generated by the stablecell lines developed with PCORON 1002-EGFP-C1-βGal-PSLD wassignificantly smaller (approximately ten-fold) than that produced bycells lines having the flexible seven amino acid linker. This isprobably due to the size of the βGal protein placing large demands uponthe transcriptional and translational machinery of the cell.

A stable cell line developed with PCORON1002-EGFP-C1-PSLD (see FIG. 13)was homogeneous (average total cell RFU 435, SD 58; n=271; see FIG. 10)in nature and provided sensitive, stable and uniform assays forinvestigating the cell cycle and for rapidly screening the effect ofagents upon the cell cycle in multiwell plate format (Tables 1 and 2;and FIG. 10).

Certain aspects of the invention disclosed hereinabove has beenpublished in Molecular Biology of the Cell (15: 3320-3332, July 2004)and electronically published as MBC in press, 10.1091/mbc.E04-03-0227 onMay 14, 2004, under the title of “Cell Cycle-dependent Regulation of aHuman DNA Helicase That Localizes in DNA Damage Foci”, the disclosure ofwhich is incorporated herein by reference in its entireties.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. Although a few exemplary embodiments ofthis invention have been described, those skilled in the art willreadily appreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention as defined inthe claims. Therefore, it is to be understood that the foregoing isillustrative of the present invention and is not to be construed aslimited to the specific embodiments disclosed, and that modifications tothe disclosed embodiments, as well as other embodiments, are intended tobe included within the scope of the appended claims. The invention isdefined by the following claims, with equivalents of the claims to beincluded therein.

1. An isolated nucleic acid construct encoding a polypeptide constructwhich comprises a detectable live-cell reporter molecule linked via apolypeptide group having a molecular mass of less than 1,000 Daltons toanother polypeptide consisting of amino acid sequence 957-1087 of SEQ IDNO:1, which amino acid sequence is the phosphorylation-dependentsubcellular localization domain of the C-terminal special control regionof helicase B (PSLD), wherein the translocation of said construct withina mammalian cell is indicative of the cell cycle position.
 2. Thenucleic acid construct of claim 1, wherein said construct additionallycomprises and is operably linked to and under the control of at leastone cell cycle independent expression control element.
 3. The nucleicacid construct of claim 2, wherein said expression control element iseither an ubiquitin C promoter or a CMV promoter.
 4. The nucleic acidconstruct of claim 1, comprising a CMV promoter, and sequences encodingPSLD and EGFP or J-Red.
 5. The nucleic acid construct of claim 1,comprising a ubiquitin C promoter, and sequences encoding PSLD and EGFPor J-Red.
 6. A vector comprising the nucleic acid construct of claim 1.7. A vector according to claim 6, wherein said vector is either a viralvector or a plasmid.
 8. A vector according to claim 7, wherein saidviral vector is an adenoviral vector or a lentiviral vector.
 9. Anisolated host cell transfected with the nucleic acid construct ofclaim
 1. 10. The host cell according to claim 9, wherein said cell is ahuman cell.
 11. A stable cell line comprising one or more of the hostcells of claim
 9. 12. The nucleic acid construct of claim 1, whereinsaid polypeptide group has a molecular mass of less than 700 Daltons.13. The nucleic acid construct of claim 1, wherein said polypeptidegroup has a molecular mass of less than 500 Daltons.
 14. The nucleicacid construct of claim 1, wherein said polypeptide group is aheptapeptide.
 15. The nucleic acid construct of claim 14, wherein saidheptapeptide isGlycine-Asparagine-Glycine-Glycine-Asparagine-Alanine-Serine (SEQ IDNO:18).
 16. The nucleic acid construct of claim 1, wherein the live-cellreporter molecule is selected from the group consisting of fluorescentprotein, enzyme reporter and antigenic tag.
 17. The nucleic acidconstruct of claim 16, wherein said fluorescent protein is selected fromthe group consisting of Green Fluorescent Protein (GFP), Enhanced GreenFluorescent Protein (EGFP), Emerald and J-Red.